Human natural killer (NK) cells comprise a small population of peripheral blood lymphocytes, phenotypically identified as CD56+/CD3- cells, that possess the unique capacity to kill virus-infected or transformed cells in a HLA-unrestricted manner. NK cell-induced lysis of cells, perhaps at sites of tumor destruction or tissue inflammation, releases intracellular contents into the extracellular space. Released purine metabolites appear to be involved in the regulation of immune function. In fact, treatment of mouse cytotoxic lymphocytes with nicotinamide adenine dinucleotide (NAD) (10 mM), but not ADP-ribose, AMP, or adenosine, resulted in the ADP-ribosylation of membrane proteins and in an inhibition of cytotoxic lymphocyte proliferation. Only a partial inhibition of cytotoxicity was observed following incubation with higher concentrations of NAD (100 mM). It was shown subsequently that the inhibition of cytotoxic lymphocyte proliferation was associated with a decrease in intracellular tyrosine phosphorylation mediated by p56. In addition, extracellular ATP affects signaling in lymphocytes by acting on P2 purinergic receptors, which open membrane ion channels or activate G protein-coupled phospholipases. In fact, ATP and P2 receptors appear to be involved in triggering apoptosis during clonal deletion of thymocyte subsets in the thymus. Extracellular adenosine, however, induces an increase in intracellular Ca2+ or cAMP by signaling through P1 purinoceptors.We hypothesized that purine metabolites could serve as feedback regulators of NK cell proliferation. NK (CD56+/CD3-) cells were incubated with IL-2 (1000 U/ml) in a 4-day thymidine uptake assay with or without 10-10,000 micromolar of nucleotides. Adenine nucleotides inhibited NK cell proliferation, with ATP = ADP > 5'-adenylylimidodiphosphate > AMP + adenosine. ADP-ribose and nicotinamide adenine dinucleotide, but not nicotinamide or UTP, caused a dose-dependent suppression of thymidine uptake. 100 micromolar ATP, a concentration that induced a maximal (80%) inhibition of thymidine uptake, did not inhibit toxicity against K562 cells. Because NK cells retained the ability to lyse K562 targets 4 days after exposure to 500 micromolar ATP or 1 millimolar adenosine, inhibition of thymidine uptake was not due to cell death. Incubation of NK cells with dibutyryl cAMP or forskolin also suppressed thymidine uptake. Cholera toxin and pertussis toxin suppressed NK cell proliferation. Pertussis toxin did not block the adenine nucleotide effects. Further, ATP, but not adenosine or other nucleotides, markedly increased intracellular cAMP in a concentration-dependent manner that was specific because CD19+ B and CD4+ T cell/cAMP did not increase. These studies demonstrate that NK proliferation is regulated through purine receptors by adenine nucleotides, which may play a role in decreasing NK cell activity. The response to adenine nucleotides is lineage-specific.